Bacterial composition

ABSTRACT

A bacterial composition that inhibits  E. coli  O157:H7 growth by as much as 93% and  Salmonella  growth by as much as 97%, together with commensurate inhibition rates against the Big-Six  Escherichia coli  strains referred to as the non-O157 STECs that include  E. coli  O121:H19;  E. coli  O45:H2;  E. coli  O103:H11;  E. coli  O145,  E. coli  O26:H11; and  E. coli  O111. The composition is constituted of various combinations of the following four unique pathogen-inhibiting bacteria: (1)  Lactobacillus animalis  strain MB101; (2)  Lactobacillus animalis  strain MB102; (3)  Enterococcus faecium  strain MB505; and (4)  Pediococcus acidilactici  strain MB902. Each of the discovered bacteria is deposited with the American Type Culture Collection (ATCC) and respectively has an individual ATCC Accession Number.

FIELD

The present disclosure relates generally to compositions and methods formanufacture and use of pathogen inhibiting bacteria.

BACKGROUND

Certain bacteria have been recognized for their capability to inhibitthe growth of certain pathogenic bacteria, and have therefore beenutilized as additives in products for which pathogenic inhibition isadvantageous. At least one example is animal feed to which bacteria aresometimes added and that have been found to improve animal efficiencyand health. Such bacterial feed additives are frequently referred to asprobiotics, as well as Direct Fed Microbials, or DFMs.

BRIEF DESCRIPTION OF THE DRAWINGS

Implementations of the present technology will now be described, by wayof example only, with reference to the attached figures, wherein:

FIG. 1 is a diagram representing pathogen inhibition by the applicationof variously composed bacterial compositions as disclosed herein; and

FIGS. 2 and 3 are tables detailing discovered pathogen inhibitionefficiencies of the bacteria and bacterial compositions disclosedherein.

DETAILED DESCRIPTION

One of the larger economic burdens facing cattle owners is the high costof rearing and/or replacing animals to maintain or increase herd size. Amajor factor contributing to the high cost of cattle replacement is theprevalence of diarrheal disease, known as scours, in livestock. Scourscauses greater than 60% of all deaths associated with pre-weaned calves,and accounts for 6.2% of total calf losses. The prevalence of scours canvary dramatically (4.3% to 52.4%) depending on herd, diet, season, or“outbreak” occurrences. Estimates of scouring rates within a herd aredifficult to obtain, though they are believed to between 15% and 35%.Nonetheless, it is agreed that diarrheal events comprise the largesthealth challenge to pre-weaned calves.

Moreover, diarrhea (scours) remains the predominant cause of mortalityamong calves. There are multiple causes of scours includingmalabsorption and improper nutrition; however, infections by bacteria,viruses, and protozoa are the primary etiological agents. It isimportant to consider that scours in calves may be due to a number ofconcurrent gastrointestinal insults by numerous pathogens.Susceptibility to acute undifferentiated diarrhea can be largelydetermined by the quantity, quality, and administration time ofcolostrum.

The costs associated with scours are difficult to estimate; however,mortality alone represents a large expense, since, at birth, a heiferhas an estimated value of $400-$600. Scours does not always result indeath, but costs associated with treatment (e.g. electrolytes,antibiotics, veterinary services and associated labor) can besignificant. In addition, animal sickness and death can negativelyimpact the morale of farm laborers and must be taken into consideration,though the financial costs of this cannot be readily quantified.

Serum immunoglobulin obtained from colostrum can offer some limitedprotection to calves from bacterial and viral infections. However, thisprotective effect begins to diminish <96 hours after birth, which couldexplain the high onset of viral scours 5-7 days following birth.Prophylactic antibiotics and vaccines administered to calves arefrequent measures used to prevent scours in calves. While antibioticadministration can be effective against bacterial infections,antibiotics are ineffective against viruses and protozoa and, in fact,they can promote the development of viral or protozoal scours bydiminishing the normal protective flora. Moreover, the use ofantibiotics is disfavored in many settings and can otherwise compromisethe health and/or value of the animal. Vaccinations can also conferprotection against scours; however, the full protective immune responsedoes not occur until after a few weeks of administration. Despite someadvances in prevention and treatment, the incidence of scours can varywildly between cattle herds.

A major factor contributing to the onset of scours in calves is thepractice of removing calves from their mother cows immediately afterbirth, and transporting them to facilities away from adult animals. Thegastrointestinal tracts of mammals, including calves, are sterile atbirth, but rapidly become colonized by microflora located near themother's vagina and anus. Other bacteria begin to establish themselveswhen the neonate comes into contact with new objects (feed, dirt, gates,fences, handlers, etc.). Prior to the current practice of removing acalf from its mother, protective microflora would become established inthe calf due to contact with the mother via licking, nursing, andgrooming. Thus, one possible avenue to reduce the incidence and severityof scours includes manipulating the bacterial flora of a calves'digestive tract.

It has long been known that a number of beneficial bacteria colonize theintestinal tracts of mammals and can promote the well-being of the host.It has also been recognized for many years that the consumption ofexogenous bacteria, often referred to as probiotics, can elicitbeneficial effects upon a host. In humans, these probiotic bacteria havebeen shown to reduce the severity and duration of rotaviral-induceddiarrhea, alleviate lactose intolerance, and enhance gastrointestinalimmune function. Traditionally, food sources such as yogurt have beenconsidered probiotic-carriers providing these health-promoting benefits.It is believed that the consumption of foods rich in probiotic bacteria,including lactic acid bacteria and bifidobacteria, leads to colonizationof the human gastrointestinal tract of humans.

The consumption of probiotics by animals used in food production canimprove their health and feed utilization efficiencies, as well asdecrease certain pathogen loads within the animal and pathogen sheadingoutside, from the animal. Probiotics work by competitive exclusion inwhich live bacterial cultures act antagonistically on specific organismsto cause a decrease in the numbers of that organism. Mechanisms ofcompetitive exclusion include production of antibacterial agents(bacteriocins) and metabolites (organic acids and hydrogen peroxide),competition for nutrients, and competition for adhesion sites on the gutepithelial surface. Lactic acid producing bacteria are generallyconsidered as food grade organisms and there are many potentialapplications of protective cultures in various foods. A number ofdifferent factors have been identified that contribute to theantibacterial activity of lactic acid producing bacteria. These bacteriaproduce different antibacterials, such as lactic acid, acetic acid,hydrogen peroxide, carbon dioxide and bacteriocins, which can inhibitpathogenic microorganisms.

A majority of bacteriocins produced by bacteria are lantibiotics orsmall hydrophobic heat stable peptides. Nisin, a lantibiotic iseffective at inhibition of Gram-positive bacteria such as Bacillus andClostridium. However, Nisin has demonstrated no effectiveness againstGram-negative bacteria. Among the small hydrophobic heat stablepeptides, pediocins are frequently encountered and possess the abilityto inhibit Listeria monocytogenes.

Lactobacillus genus includes the most prevalently administered probioticbacteria. Lactobacillus is a genus of more than 25 species ofgram-positive, catalase-negative, non-sporulating, rod-shaped organisms.Lactobacillus species ferment carbohydrates to form lactic acid.Lactobacillus species are generally anaerobic, non-motile, and do notreduce nitrate. Lactobacillus species are often used in the manufactureof food products including dairy products and other fermented foods.Lactobacillus species inhabit various locations including thegastrointestinal tracts of animals and intact and rotting plantmaterial. Lactobacillus strains appear to be present in thegastrointestinal tract of approximately 70% of humans that consume aWestern-style diet. The number of Lactobacillus cells in neonates isapproximately 105 colony forming units (CFU) per gram CFU/g of feces.The amount in infants of one month and older is higher, ranging from 106to 108 CFU/g of feces.

Lactic acid and products containing lactic acid enhance gains in thestarting period of feedlot cattle (first 28 days) and reduce liverabscesses when administered during the transition from a primarilyroughage diet of grass to a feedlot diet including more grains. Certainstrains of Lactobacillus acidophilus have been isolated which restoreand stabilize the internal bacterial balance of animals. Some strainsdemonstrate a greater propensity to adhere to the epithelial cells ofsome animals which would increase their ability to survive, initiate andmaintain a population within an animal intestine. Thus, the primary modeof action as previously understood relative to Lactobacillus acidophilusoccurs post-ruminally.

The most common method used today to control pathogenic populations inlivestock is through the use of antibacterial compounds. While these areeffective for short-term treatments, prolonged application ofantibacterial compounds leads to the evolution of antibiotic resistancein the pathogenic organisms. The widespread occurrence of antibioticresistant microorganisms is well known; two examples are methicillinresistant Staphylococcus aureus (MRSA) and vancomycin resistantenterococci (VRE). Bacteria are remarkably adaptable to deleteriousenvironments with their abilities to rapidly reproduce and modify theirgenetic content. Thus, it is inevitable that after prolonged applicationof any method that disrupts or kills bacteria, a population that isrecalcitrant to its effects will eventually arise. It is not uncommonnow in the veterinary environment that doctors often resort to usingmultiple antibiotics concurrently or in succession to eradicatepathogenic organisms.

As with antibiotics, bacteria can also become resistant to otherbiological treatments. For example, bacteriophages are able to reducepathogen populations, but inevitably, a fraction of the targetedbacterial population is not affected. This small sub-population thenrapidly reproduces and attains sizable population numbers.

Similar circumstances have been seen with the application of probioticbacteria that are meant to inhibit or reduce the numbers of pathogenicbacteria within a gastrointestinal system. Some researchers havecommented that significantly better animal performance and pathogenreductions were seen in treated animals early in their experiments, butthe beneficial effects were no longer statistically different afterprolonged application of the probiotic product. It is possible that thetarget populations were initially affected, but prolonged usage of theprobiotic product led to the selection of bacterial populations thatwere not influenced by the application of the product. According to thisdisclosure, the adaptation of pathogens to probiotic treatment can beavoided with the inclusion of multiple strains of bacteria.

There are numerous advantages provided by the inclusion of multiplestrains of bacteria in a bacterial composition such as that which isdisclosed herein. These advantages, whether working independently orconcurrently, support the superiority of the presently disclosedbacterial composition and the enhanced benefits for an animal to whichit is administered.

Different bacteria strains utilize certain nutrients more efficientlythan others. The ability to use available nutrients in a gut environmentis necessary for the bacteria to produce antibacterial compounds or tobeneficially affect the host GI system. However, the nutrientavailability is constantly changing because of animal behavior,different foods consumed, antibiotic use, energy requirements, or healthof the animal. These fluctuations allow different bacteria toproliferate while other bacterial populations diminish.

The use of different bacterial strains also produces different bacterialmetabolites. Different metabolites have different effects uponpathogenic populations. Lactic acid is a powerful antibacterial agentagainst some pathogens, while propionic acid is more effective againstother populations. It should also be considered that just as metabolitesproduced from cells in bacterial products affect GI populations,endogenous microorganisms produce chemicals that can be inhibitory tosome bacteria strains. The present inclusion of different strains in adirect fed feed supplement increases the likelihood that the productwill have a positive effect.

Additionally, the production of bacteriocins influences bacterialpopulations. There is a large diversity of bacteriocins that targetspecific bacteria populations. Thus, a bacterial product that containsmultiple strains will produce multiple bacteriocins and target differentgroups of pathogenic populations. Conversely, the intestinal tractcontains a large diversity of bacteriocin producing bacteria. While someof the produced bacteriocins can affect one of the included strains, itis unlikely to affect all of the included microorganisms.

Another benefit of the presently disclosed multi-strain bacterialcomposition when utilized as a direct fed feed additive is its abilityto target more than one pathogen population for inhibition. Bacterialpathogens are very diverse and require different methods to reduce oreliminate their populations. Thus, a product containing differentpathogen inhibiting bacteria that are able to effect differentpathogenic populations will result in an overall healthier animal andherd.

Different microorganisms positively influence the gastrointestinalsystem through different mechanisms. Therefore, including bacteria thatwork through different modes will result in a superior product. Onestrain may reduce pathogen populations, while another has animmunostimulative effect, while another produces micronutrientsessential for the host. Interestingly, multiple strains can also providesynergistic effects upon the host or pathogen inhibition abilities. Onestrain alone may not be able to reduce certain populations, but thecombination of two or more different strains working through differentmechanisms can reduce pathogen populations.

Additionally, the use of multiple beneficial microorganisms can helpovercome bacteriophages that infect and kill bacteria. Bacteriophagesare very common in gastrointestinal systems and have profound effectsupon the bacterial community. Bacteriophages require specific sites on acell to bind and infect. Thus, by including multiple microorganisms in aproduct, the greater the likelihood that at least some populations fromthe product will evade bacteriophage attack and elicit beneficialeffects upon the bacterial community and host.

Certain examples in the present disclosure concern a method ofinhibiting or reducing a population of pathogenic bacteria in, on and/oroutside the animal. In one aspect, the disclosed compositions reduce apopulation of pathogenic bacteria in the gastrointestinal tract of theanimal.

Other examples concern the inhibition or reduction of a population ofpathogenic bacteria by providing to the animal a composition containingthe multiple probiotic bacteria described herein.

In the examples of the present disclosure wherein an administration ofpathogen inhibiting bacteria is contemplated, the number of bacteria(concentration) per administration can be any amount capable ofproviding some inhibition or reduction of a population of pathogenicbacteria. In specific embodiments, the number of bacteria peradministration is between 1×10³ and 1×10⁹ bacteria. Preferably, thenumber of bacteria in an administration is approximately 1×10⁶ bacteria.

The administrations can be timed such that a series of administrationsof the composition or its separate components is separated by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 17, 19, 20, 21, 22 or23 hours or 1, 2, 3, 4, 5, or 6 days or 1, 2 or 3 weeks or 1 month orsome duration in between. In some specific embodiments, theadministrations are daily.

Among others, administration of the bacterial composition to the animalcan be oral, nasal, topical, rectal, and via injection. In certainembodiments, the administrations are oral administrations. Inembodiments wherein the administrations are oral administrations, thecomposition comprising probiotic bacteria can be mixed with animal feedor mixed with animal drinking water. In such embodiments, thecomposition or compositions can be formulated as a liquid formulationfor administration, or as a freeze dried formulation, or as a gelformulation or as a spore formulation.

Additional steps in inhibiting or reducing the population of pathogenicbacteria in an animal include assessing the presence of pathogenicbacteria in the gastrointestinal tract of the animal betweenadministrations. In more specific embodiments, the animal is assessedfor the presence of pathogenic bacteria, for strains of pathogenicbacteria, species of pathogenic bacteria and number (concentration) ofpathogenic bacteria present. In specific embodiments, this assessment isdone by examining the feces of the animal.

In the context of the present disclosure, the terms “substantially” and“about” are defined to be essentially conforming to the particularquantity, concentration, dimension, shape or other thing that“substantially” or “about” modifies, such that the so describedcharacteristic need not be exact, but within reasonable tolerances. Theterms “comprising,” “including” and “having” (and variants thereof) areused interchangeably in this disclosure. The terms “comprising,”“including” and “having” mean to include, but not necessarily be limitedto the things so described.

It should be understood that this disclosure is not limited toparticular compositions or biological systems, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferred methodsand materials are now described.

In this specification and the claims that follow, reference will be madeto a number of terms which can be considered to have the followingmeanings: “inhibit” and/or “reduce” or other forms of the words andtheir synonyms, such as “reducing” or “reduction,” refer to slowing thegrowth of the so-referenced pathogen and/or lowering its incidence. Itis understood that this is typically in relation to some standard orexpected value, in other words it is relative, but that it is not alwaysnecessary for the standard or relative value to be referred to. Forexample, “reduces the population of bacteria” in certain instances referto lowering the amount of bacteria relative to a standard or a control.“Inhibit” and “inhibition” refer to slowing or deterring pathogengrowth, including pathogenic bacterial growth that would have otherwiseoccurred except for the provision of the characterized deterrent.

By “treat” or other forms of the word, such as “treated” or “treatment,”means to administer a composition or to perform a method in order toreduce, prevent, inhibit, break-down, or eliminate a particularcharacteristic or event.

The term “viable cell” means a microorganism, and in particular,bacteria that are alive and capable of regeneration and/or propagation,while in a vegetative, frozen, preserved, or reconstituted state.

The term “viable cell yield” or “viable cell concentration” refers tothe number of viable cells in a liquid culture, concentrated, orpreserved state per a unit of measure, such as liter, milliliter,kilogram, gram or milligram.

The term “cell preservation” refers to a process that takes a vegetativecell and preserves it in a metabolically inert state that retainsviability over time. As used herein, the term “product” refers to abacterial composition that can be blended with other components andcontains specified concentration of viable cells that can be sold andused.

As used herein, the terms “microorganism” or “microbe” refers to anorganism of microscopic size. The definition of microorganism hereinincludes bacteria, Archaea, single-celled Eukaryotes (protozoa, fungi,and ciliates), and viral agents (viruses). The term “microbial” is usedherein to describe processes or compositions of microorganisms, thus a“microbial-based product” is a composition that includes microorganisms,cellular components of the microorganisms, and/or metabolites producedby the microorganisms. Microorganisms can exist in various states andoccur in vegetative, dormant, or spore states. Microorganisms can alsooccur as either motile or non-motile, and may be found as planktoniccells (unattached), substrate affixed cells, cells within colonies, orcells within a biofilm.

The term “bacteria” refers to one-celled organisms that are Prokaryotesin that their genetic material, or DNA, is not enclosed in a nucleus.

The term “prebiotic” refers to food ingredients that are not readilydigestible by endogenous host enzymes and confer beneficial effects onan organism that consumes them by selectively stimulating the growthand/or activity of a limited range of beneficial microorganisms that areassociated with the intestinal tract.

The term “probiotic” refers to one or more live microorganisms thatconfer beneficial effects on a host organism. Benefits derived from theestablishment of probiotic microorganisms within the digestive tractinclude reduction of pathogen load, improved bacterial fermentationpatterns, improved nutrient absorption, improved immune function, aideddigestion and relief of symptoms of irritable bowel disease and colitis.

The term “synbiotic” refers to a composition that contains bothprobiotics and prebiotics. Synbiotic compositions are those in which theprebiotic compound selectively favors the probiotic microorganism.

The term “gastrointestinal tract” refers to the complete system oforgans and regions that are involved with ingestion, digestion, andexcretion of food and liquids. This system generally consists of, butnot limited to, the mouth, esophagus, stomach and or rumen, intestines(both small and large), cecum (plural ceca), fermentation sacs, and theanus.

The term “pathogen” refers to any microorganism that produces a harmfuleffect and/or disease state in a human or animal host.

The term “fermentation” refers to a metabolic process performed by anorganism that converts one substrate to another in which the cell isable to obtain cellular energy, such as when an organism utilizesglucose and converts it to lactic acid or propionic acid. Many of theend-substrates formed in fermentation processes are volatile fattyacids.

The term “volatile fatty acids” (VFAs) refers to short-chain fatty acidscontaining six or fewer carbon atoms and at least one carboxyl group.Some examples of VFAs include, but are not limited to: lactic acid,acetic acid, propionic acid, butyric acid, isobutyric acid, valericacid, and isovaleric acid, which are products of bacterial fermentationwithin the digestive tracts of animals. Volatile fatty acids can beabsorbed through the intestines of animals and used as an energy orcarbon source. Bacteria produce VFAs based on available substrates andalso rely upon VFAs for energy and carbon sources.

The term “lactic acid” refers to a byproduct of glucose fermentationresulting in a three-carbon acid with the chemical formula C₃H₆O₃. Thisincludes, but is not limited to, lactic acid derived from specificstrains of bacteria or lactic acid derived from other types oforganisms. Lactic acid can be microbialstatic, microbialcidal,bacteriostatic, bacteriocidal or bacteriolytic; these concepts are knownto skilled persons. “Lactic acid producing” refers to any organism thatgenerates lactic acid.

The term “bacteriocin(s)” refers to (poly) peptides and proteins thatinhibit one or more bacterial species. This includes, but is not limitedto, (poly) peptides or proteins that were derived from specific strainsof bacteria or (poly) peptides that are derived from other types oforganisms.

The bacteriocin can be microbialstatic, microbialcidal, bacteriostatic,bacteriocidal, or bacteriolytic; these concepts are known to skilledpersons. For the treatment of produce and other food products thebacteriocin is preferably microbialcidal or bacteriocidal. “Bacteriocinproducing” in certain instances refer to any organism that generatesbacteriocins.

As used herein, “hydrogen peroxide” refers to a byproduct of oxygenmetabolism that has the chemical formula H₂O₂. This includes, but is notlimited to, hydrogen peroxide derived from specific strains of bacteriaor hydrogen peroxide derived from other types of organisms. Hydrogenperoxide can be microbialstatic, microbialcidal, bacteriostatic,bacteriocidal or bacteriolytic; these concepts are known to skilledpersons. “Hydrogen peroxide-producing” refers to any organism thatgenerates hydrogen peroxide.

As used herein, the term “synergistic” refers to a property wherein thecombined result of two effects is greater than would be expected if thetwo effects were added together. The term “synergistically” is used todescribe a synergistic effect.

As used herein, the phrase “foregut fermentor” refers to an animalhaving an anatomical compartment in the alimentary canal that ispositioned anterior to the stomach that is used for bacterialfermentation and digestion of ingested materials. Ruminal fermentors areconsidered foregut-fermenting organisms.

As used herein, the phrase “ruminal fermentor” or “rumen fermenting”refers to an animal having a large, multi-compartmented section of thedigestive tract, called a rumen, which is positioned between theesophagus and the anus. Rumen are very complex ecosystems that supportbacterial fermentation of cellulose, plant matter, and other ingestedmaterials. Ruminal-fermentors may also be termed “cranial fermentors” or“ruminants”. Some examples of rumen-fermenting organisms include cattle,sheep, goats, camels, llama, bison, buffalo, deer, wildebeest andantelope.

As used herein, the phrase “hindgut fermentor” refers to an animalhaving a complex large intestine that may or may not include specializedfermentation chambers that can include a cecum or blind sac, that ispositioned posterior to the stomach in the alimentary canal. Cecalfermentors and intestinal fermentors are both consideredhindgut-fermenting organisms.

As used herein, the phrase “cecal fermentor” refers to an animal havinga complex large intestine that includes a cecum or a blind sac along thedigestive tract. The cecum of a cecal fermentor forms a distinctchamber, which is the primary site of bacterial fermentation ofcellulose, plant matter, or other ingesta. A cecal-fermentor may also bereferred to as “caudal fermentor”. Cecal-fermentors include horses,elephants, rabbits, mice, rats, guinea pigs and the like.

As used herein, the term “intestinal fermentor” refers to an animal thatdoes not primarily rely upon bacterial fermentation of ingesta in arumen or large cecum. In the digestive tracts of intestinal fermentors,bacterial fermentation occurs primarily within the large intestine orcolon. Intestinal fermentors include chickens, pigs, humans and thelike.

As used herein, the term “monogastric” refers to an animal having asingle, simple (single chambered) stomach. Typically, cecal fermentorsand intestinal-fermentors are monogastric animals. Some examples ofmonogastric animals include horses, chickens, pigs, humans and the like.

As used herein, the term “polygastric” refers to an animal having amultiple, complex (multi-chambered) stomachs. Ruminal fermentors arepolygastric animals.

As used herein, the phrase “pre-gastric fermentation” refers tobacterial fermentation that occurs before the food reaches a ‘true’stomach, which is generally the site of gastric acid and digestiveenzyme secretion. Ruminants are pre-gastric fermentors.

As used herein, the phrase “post-gastric fermentation” refers tobacterial fermentation that occurs after food passes through a stomach,which is generally the site of gastric acid and digestive enzymesecretion. Hindgut fermentors, including cecal fermentors and intestinalfermentors, utilize post-gastric fermentation.

As used herein, the term “herbivore” refers to an animal thatexclusively consumes plant material.

As used herein, the term “omnivore” refers to an animal that consumesboth plant and animal material.

As used herein, the term “carnivore” refers to an animal thatexclusively consumes animal material.

As used herein, “digesta” refers to food or any other material thatenters the alimentary canal and undergoes, completely or partially,through the process of being digested or broken down into smallercomponents.

FIG. 1 depicts and the present specification discloses a novelcomposition variously comprised of multiple bacteria strains. Thecomposition can be added to products to inhibit pathogen growth and/orreduce pathogen presence in downstream production processes, productstorage and/or product utilization. Among other uses, the compositioncan be utilized in washes, dips and the like for reducing pathogen loadand inhibiting pathogen growth; for instance, in and on food products.Surfaces such as countertops, refrigerators and food preparationsurfaces can also be treated, with special benefits being provided toporous surfaces into which the composition can be absorbed.

The presently disclosed compositions are particularly advantageous wheningested as a probiotic supplement or utilized as a direct fed bacterialfeed additive that provides beneficial effects to all types of animals,including amphibians, birds, fish, invertebrates, reptiles and mammals,including fermentors, cecal fermentors and intestinal fermentors. In oneembodiment, the probiotic formulation supplemented with prebioticcompounds is fed to ruminal fermentors to reduce scours events andimprove animal health. Ruminal fermentors that can benefit from thepresent disclosure include but are not limited to: cattle, sheep, goats,camels, llama, bison, buffalo, deer, wildebeest, antelope, and any otherpre-gastric fermentor.

In another embodiment, the probiotic formulation supplemented withprebiotic compounds is fed to cecal fermentors to reduce scours eventsand improve animal health. Cecal fermentors that can benefit from thisdisclosure include but are not limited to: horses, ponies, elephants,rabbits, hamsters, rats, hyraxes, guinea pigs, and any otherpost-gastric fermentor that using the cecum as the primary location offermentative digestion. In another embodiment, the probiotic formulationsupplemented with prebiotic compounds is fed to intestinal fermentors toreduce scours events and improve animal health. Intestinal fermentorsthat can benefit from said disclosure include but are not limited to:humans, pigs, chickens, and other post-gastric fermentor using the largeintestine as the primary location of fermentative digestion. In eachcase, the composition is packaged in a format that ensures survival ofboth the probiotic and prebiotic components into the gastrointestinalsystem of the animal.

In one aspect, the present disclosure provides a novel composition thatreduces animal mortality and morbidity and reduces pathogen load in theanimal, as well as pathogen sheading in its feces. Administration of thebacteria-based compositions to the animal also can improve animal healthand/or productivity by way of increased feed efficiency. It alsoprovides a composition that will be used once, periodically or on acontinual basis to reduce the incidence and severity of scours and/orimprove animal health. The composition is durable and easy to apply toanimal feed or other easily ingestible materials.

The novel compositions disclosed comprise specially selected bacteria,at least some of which can produce lactic acid which inhibits the growthof pathogenic organisms during digestive fermentation. The compositioncan comprise a mixture of lactic acid producing bacteria. In someembodiments, the composition is mixed with colostrum or milk replacers.In other embodiments the mixture is applied to milk or wateradministered to calves. Animals can be treated with a combination ofviable microorganisms with prebiotic compounds to improve animalefficiency and/or health. Additive, or more preferably super-additive ormore preferably synergistic effects can be achieved with theadministration of two or more bacteria species and/or strains. Animalscan be treated once, multiple times, or therapeutically on a dailybasis.

In one aspect, various compositional examples of this disclosure includebacterial combinations that are pathogen inhibitive in that thecombination inhibits pathogen growth when added thereto. The speciallyselected bacteria can be of different species (for example, L. animalisversus Enterococcus faecium), or they may be of the same species (forexample, L. animalis and L. animalis) but different strains (forexample, MB101 and MB102) within the same species (L. animalis). That isto say, the composition may contain multiple species and/or multiplestrains. For example, two, three, four, five, six, and so on differentbacterial strains can be included. The use of multiple types of thespecially selected bacterial strains lead to a superiorly reliableproduct for maintaining or improving animal health or inhibiting,decreasing or eliminating the presence of pathogenic bacteria. As anexample, each of the bacteria in the composition can have superiorsurvival characteristics under various conditions likely to beencountered during storage, transport and/or administration. In that wayit is better assured that viable bacteria will reach the target (animal,object and the like), regardless of whether one or more abusiveconditions (such as overheating) have been encountered aftermanufacture.

More specifically, among others, four different pathogen inhibitingbacteria strains have been discovered and are variously included in theinstantly disclosed bacterial compositions. Each strain was deposited onNov. 7, 2014 with the American Type Culture Collection (ATCC) located atATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110 andthe corresponding certificates issued on Nov. 25, 2014 carrying thetitle of Budapest Restricted Certificate of Deposit Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure International Form Receipt in the Case of anOriginal Deposit Issued Pursuant to Rule 7.3 and Viability StatementIssued Pursuant to Rule 10.2. The deposits, their taxonomicdescriptions, dates of deposit and accession numbers have been certifiedas follows:

ATCC Strain Taxonomic Date Accenssion Name Description Deposited NumberMB505 Enterococcus faecium Nov. 7, 2014 PTA-121709 MB101 Lactobacillusanimalis Nov. 7, 2014 PTA-121710 MB102 Lactobacillus animalis Nov. 7,2014 PTA-121711 MB902 Pediococcus acidilactici Nov. 7, 2014 PTA-121712

In FIG. 2 of the present disclosure, specific compositional combinationsof the discovered strains are disclosed together with their derivedpathogen inhibition efficacies specified in terms of percent pathogengrowth inhibition determined using the processes described below. Thederived pathogen inhibition efficacies of the individual strains areshown in FIG. 3.

Experiments demonstrating the ability of the strains to inhibit thegrowth of pathogenic organisms were performed using the followingmethod. A “control” tube was prepared containing an amount of modifiedMRS medium to which an amount { concentration at 1×10⁶ CFU/ml} of asingle strain pathogenic bacteria {E. coli or Salmonella, for example}was added. A “challenged” tube was prepared that contained the sameamount of the modified MRS medium to which the same amount of the singlestrain pathogenic bacteria was added as in the control tube, togetherwith an equal total amount {concentration at 1×10⁶ CFU/ml} ofchallenging bacterium strain(s) as the pathogenic bacteria. The tubeswere placed into a water bath at 37° C. and incubated for six hours.After incubation, each tube was serially diluted and 100 microliters ofits content spread onto Luria-Bertani plates to enumerate the number ofviable pathogen cells. The percent pathogen growth inhibition was thencalculated as: the difference between the amount of viable pathogencells in the challenged tube versus the control tube, divided by theamount of viable pathogen cells in the control tube, and then multipliedby 100.

Similar illustrative procedures are disclosed and described in U.S.Patent Publication 2011-0189132, the entirety of which is herebyincorporated herein by reference.

New compositions that deliver pathogen growth inhibition are variouslydisclosed in FIG. 2, including those that comprise at least two of anyof the following four bacteria: (1) Lactobacillus animalis strain MB101having ATCC Accession Number PTA-121710; (2) Lactobacillus animalisstrain MB102 having ATCC Accession Number PTA-121711; (3) Enterococcusfaecium strain MB505 having ATCC Accession Number PTA-121709; and (4)Pediococcus acidilactici strain MB902 having ATCC Accession NumberPTA-121712.

Exemplarily, a composition is described that includes (1) Lactobacillusanimalis strain MB101 having ATCC Accession Number PTA-121710 and (2)Lactobacillus animalis strain MB102 having ATCC Accession NumberPTA-121711. The composition demonstrated the following pathogeninhibition percentages: 91.9% inhibition of E. coli O157:H7; 91.1%inhibition of Salmonella typhimurium and 95.3% inhibition of Salmonellaenteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB101 having ATCC Accession Number PTA-121710; (2)Lactobacillus animalis strain MB102 having ATCC Accession NumberPTA-121711; and (3) Enterococcus faecium strain MB505 having ATCCAccession Number PTA-121709. The composition demonstrated the followingpathogen inhibition percentages: 90.9% inhibition of E. coli O157:H7;90.0% inhibition of Salmonella typhimurium and 94.0% inhibition ofSalmonella enteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB101 having ATCC Accession Number PTA-121710; (2)Lactobacillus animalis strain MB102 having ATCC Accession NumberPTA-121711; (3) Enterococcus faecium strain MB505 having ATCC AccessionNumber PTA-121709; and (4) Pediococcus acidilactici strain MB902 havingATCC Accession Number PTA-121712. The composition demonstrated thefollowing pathogen inhibition percentages: 93.1% inhibition of E. coliO157:H7; 97.4% inhibition of Salmonella typhimurium and 96.0% inhibitionof Salmonella enteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB101 having ATCC Accession Number PTA-121710 and (2)Enterococcus faecium strain MB505 having ATCC Accession NumberPTA-121709. The composition demonstrated the following pathogeninhibition percentages: 92.1% inhibition of E. coli O157:H7; 95.8%inhibition of Salmonella typhimurium and 95.4% inhibition of Salmonellaenteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB101 having ATCC Accession Number PTA-121710; (2)Enterococcus faecium strain MB505 having ATCC Accession NumberPTA-121709; and (3) Pediococcus acidilactici strain MB902 having ATCCAccession Number PTA-121712. The composition demonstrated the followingpathogen inhibition percentages: 85.7% inhibition of E. coli O157:H7;87.0% inhibition of Salmonella typhimurium and 90.5% inhibition ofSalmonella enteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB101 having ATCC Accession Number PTA-121710 and (2)Pediococcus acidilactici strain MB902 having ATCC Accession NumberPTA-121712. The composition demonstrated the following pathogeninhibition percentages: 92.7% inhibition of E. coli O157:H7; 96.3%inhibition of Salmonella typhimurium and 95.4% inhibition of Salmonellaenteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB101 having ATCC Accession Number PTA-121710; (2)Lactobacillus animalis strain MB102 having ATCC Accession NumberPTA-121711; and (3) Pediococcus acidilactici strain MB902 having ATCCAccession Number PTA-121712. The composition demonstrated the followingpathogen inhibition percentages: 90.8% inhibition of E. coli O157:H7;89.7% inhibition of Salmonella typhimurium and 94.0% inhibition ofSalmonella enteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB102 having ATCC Accession Number PTA-121711 and (2)Enterococcus faecium strain MB505 having ATCC Accession NumberPTA-121709. The composition demonstrated the following pathogeninhibition percentages: 86.3% inhibition of E. coli O157:H7; 87.1%inhibition of Salmonella typhimurium and 90.8% inhibition of Salmonellaenteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB102 having ATCC Accession Number PTA-121711; (3)Enterococcus faecium strain MB505 having ATCC Accession NumberPTA-121709; and (4) Pediococcus acidilactici strain MB902 having ATCCAccession Number PTA-121712. The composition demonstrated the followingpathogen inhibition percentages: 91.6% inhibition of E. coli O157:H7;90.7% inhibition of Salmonella typhimurium and 94.8% inhibition ofSalmonella enteriditis.

Another composition is described that includes (1) Lactobacillusanimalis strain MB102 having ATCC Accession Number PTA-121711 and (2)Pediococcus acidilactici strain MB902 having ATCC Accession NumberPTA-121712. The composition demonstrated the following pathogeninhibition percentages: 84.4% inhibition of E. coli O157:H7; 82.7%inhibition of Salmonella typhimurium and 87.9% inhibition of Salmonellaenteriditis.

Another composition is described that includes (1) Enterococcus faeciumstrain MB505 having ATCC Accession Number PTA-121709 and (2) Pediococcusacidilactici strain MB902 having ATCC Accession Number PTA-121712. Thecomposition demonstrated the following pathogen inhibition percentages:86.5% inhibition of E. coli O157:H7; 87.3% inhibition of Salmonellatyphimurium and 91.7% inhibition of Salmonella enteriditis.

As shown in FIG. 3, Lactobacillus animalis strain MB101 having ATCCAccession Number PTA-121710 alone demonstrated the following pathogeninhibition percentages: 93.9% inhibition of E. coli O157:H7; 97.9%inhibition of Salmonella typhimurium; 97.2% inhibition of Salmonellaenteriditis; 96.3% inhibition of E. coli O121:H19; 94.4% inhibition ofE. coli O45:H2; 92.8.% inhibition of E. coli O103:H11; 93.0% inhibitionof E. coli O145, 91.9% inhibition of E. coli O26:H11; and 85.0%inhibition of E. coli O111.

Individually, Lactobacillus animalis strain MB102 having ATCC AccessionNumber PTA-121711 demonstrated the following pathogen inhibitionpercentages: 90.1% inhibition of E. coli O157:H7; 87.8% inhibition ofSalmonella typhimurium; 93.9% inhibition of Salmonella enteriditis;91.2% inhibition of E. coli O121:H19; 90.8% inhibition of E. coliO45:H2; 94.7.% inhibition of E. coli O103:H11; 87.4% inhibition of E.coli O145, 89.3% inhibition of E. coli O26:H11; and 85.9% inhibition ofE. coli O111.

Individually, Enterococcus faecium strain MB505 having ATCC AccessionNumber PTA-121709 demonstrated the following pathogen inhibitionpercentages: 88.2% inhibition of E. coli O157:H7; 87.8% inhibition ofSalmonella typhimurium; 92.3% inhibition of Salmonella enteriditis;86.6% inhibition of E. coli O121:H19; 70.1% inhibition of E. coliO45:H2; 88.2.% inhibition of E. coli O103:H11; 87.5% inhibition of E.coli O145, 86.9% inhibition of E. coli O26:H11; and 89.8% inhibition ofE. coli O111.

Individually, Pediococcus acidilactici strain MB902 having ATCCAccession Number PTA-121712 demonstrated the following pathogeninhibition percentages: 85.2% inhibition of E. coli O157:H7; 68.3%inhibition of Salmonella typhimurium; 87.5% inhibition of Salmonellaenteriditis; 88.2% inhibition of E. coli O121:H19; 88.6% inhibition ofE. coli O45:H2; 91.5.% inhibition of E. coli O103:H11; 86.0% inhibitionof E. coli O145, 87.3% inhibition of E. coli O26:H11; and 86.4%inhibition of E. coli O111.

Each of (1) Lactobacillus animalis strain MB101 having ATCC AccessionNumber PTA-121710; (2) Lactobacillus animalis strain MB102 having ATCCAccession Number PTA-121711; (3) Enterococcus faecium strain MB505having ATCC Accession Number PTA-121709; and (4) Pediococcusacidilactici strain MB902 having ATCC Accession Number PTA-121712inhibits the growth of E. coli O157:H7, Salmonella typhimurium;Salmonella enteriditis; and the Big-Six Escherichia coli strains(referred to as the non-O157 STECs) that include E. coli O121:H19; E.coli O45:H2; E. coli O103:H11; E. coli O145, E. coli O26:H11; and E.coli O111, albeit some more effectively than others.

As an example, and as disclosed above, the constituent bacteria areprovided in equal amounts and sum to the same total amount of inhibitingbacteria as pathogenic bacteria that are initially mixed together,before incubation, and inhibition is measured.

Certain aspects of the disclosure contemplate a carrier formulation forthe bacterial combinations. The carrier may be any number of differentpercentages (weight per weight, weight per volume, or volume per volume)of the final product. The carrier can comprise any amount of about99.9%, about 95%, about 90%, about 80%, about 70%, about 60% about 50%,about 40%, about 30% and so on. The remaining composition can alsoinclude other carriers such as lactose, glucose, sucrose, salt,cellulose and the like. In specific aspects of the disclosure, thecarrier may be 50% or more of the total product. The carrier can betailored for the target animal; for instance the carrier can be tailoredfor feedlot housed cattle, dairy cattle or calves, among others,depending on the particular needs of the target.

Another means of altering intestinal flora is through the consumption ofprebiotics. Prebiotics are food ingredients that are not readilydigestible by endogenous mammalian enzymes and that selectivelystimulate the growth and activity of selected groups of intestinalmicroorganisms that confer beneficial effects upon their host.Typically, it is probiotic microorganism populations that benefit fromthe presence of prebiotic compounds. Prebiotics can consist ofoligosaccharides and other small molecules that serve as metabolicsubstrates for growth of probiotic microorganisms. While many diversemicroorganisms inhabit the intestinal tract of a host organism,prebiotic compounds are only utilized by the probiotic microorganismsand lead to selective enhancement of probiotic growth. As an example,galacto-oligosaccharides (GOS) can be used to increase the weight ofcalves and reduce the occurrence of scours, as well as reduce pathogenicpopulations. Furthermore, fructo-oligosaccharides (FOS) can be used as afiber source.

Since prebiotics can serve as a metabolic substrate for the growth ofprobiotics, synergistic effects are obtainable when administeredsubstantially contemporaneously. It has been found that the inclusion ofcertain prebiotics improves the survival of certain probioticmicroorganism because it ensures the presence of a readily availablesubstrate for that probiotic microorganism and reduces substratecompetition for nutrients with other microorganisms. A term that may beused to describe formulations that include both prebiotics andprobiotics is “synbiotics.”

Certain direct-fed microbials (DFM) mode of action require cells to beviable to be beneficial to a host. Various factors can affect theviability of bacteria. For use as a probiotic, a lactic acid producingbacteria needs to be able remain viable during processing and storageprotocols such as centrifugation, filtration, fermentation, freezedrying or lyophilization in which the bacteria may be subjected tofreezing, high pressure, and high temperature. However, without adequatepreservation many bioactive materials comprising bacterial cultures aresensitive to degradation, loss of activity, and/or viability in aqueoussolutions, particularly at ambient or higher temperatures. Accordingly,bioactive materials comprising bacterial cultures often requirerefrigeration or have short shelf lives under ambient conditions.

When bioactive materials comprising bacteria are dried, they are alsopreserved in a concentrated form. These concentrated materials oftenneed to be diluted to be disseminated in an efficient manner and at aneffective dose. The diluting materials, commonly referred to ascarriers, may comprise many different types of materials depending uponthe product that requires dilution. Common carriers for biologicalmaterials include: cellulose, sugar, glucose, lactose, whey powder, orrice hulls. However, such carriers may not provide protection againstloss of viable cells when exposed to temperatures above freezing.Products are often exposed to temperatures above freezing duringmanufacture, processing, packaging, shipment, and storage of theproducts.

Other carriers pose problems for delivery and application of products.Some carriers are essentially insoluble in aqueous solutions, thusmaking it difficult to apply the product in field operations.

Additionally, many probiotic bacteria are administered to animals inorder to control or reduce pathogenic bacterial populations. Some of thedescribed carriers (e.g. lactose, glucose, or whey powder) are readilyutilized by these pathogens as a nutrient source, thus removingnutrients and potentially exacerbating the problem.

In certain aspects of this disclosure, the prebiotic may be anyprebiotic. In still further aspects of the disclosure the prebiotic maybe fructo-oligosaccharides (FOS), inulins, isomalto-oligosaccharides,gentio-oligosaccharides, lactilol, lactosucrose, lactulose, xylosucrose,glycosylsucrose, pyrodextrins, soybean oligosaccharides,galacto-oligosaccharides (GOS), transgalactose-oligosaccharides,xylo-oligosaccharides (XOS), Malto-oligosaccharides (MOS), galactomannansuch as guar gum, locust bean gum, konjacor xanthan gum, pentosan,beta-glucan, arabinan, galactan, palantinose-oligosaccharides,gluco-oligosaccharides, cyclo-inulo-oligosaccharides,chito-oligosaccharides, agaro-oligosaccharides, neo-oligosaccharides,garo-oligosaccharides, pectins and pectic polysaccharides, etc. Inparticular aspects of the disclosure the carrier formulation includesfructo-oligosaccharides.

The carrier and composition can also have defined properties, such assolubility/insolubility in water or solubility/insolubility in fat andthe like.

Other chemicals or materials principally used for the reduction orabsorption of moisture may also be included. These may include, but arenot limited to: calcium stearate, sodium aluminosilicate, silica,calcium carbonate, zeolite, bicarbonates, sodium sulfate, silicondioxide, or ascorbic acid.

Other chemicals or materials principally used for the reduction orabsorption of oxygen may also be included. These may include, but arenot limited to, iron oxides, ascorbic acid, sodium sulfide, and silicamaterials.

The bacteria mixed with the carrier can be stored in a pouch or bagfabricated from various materials, a bottle fabricated from a variety ofmaterials, a capsule, a box, or other storage container. The compositionmay also be applied onto a variety of foods including, but not limitedto, meats, vegetables, fruits, processed foods, and others for thepurpose of pathogen inhibition. The composition can also be packaged asa probiotic supplement for human consumption, particularly whencapsulized or made into tablets.

Preservation methods for the bacteria can include a process of freezing,freeze-drying and/or spray-drying. In certain aspects, the preservedbacteria contain a viable cell concentration of 1×10⁸ to 5×10¹² cfu/g.Still further, in certain aspects the concentrations range from 5×10¹⁰cfu/g to 5×10¹³ cfu/g of bacteria.

In certain instances, a bacterial formulation for administration to asubject or a surface or other target can include a preservation matrix,which contains and preserves the bacterial culture. Such a matrix mayinclude a biologically active binding agent, an antioxidant, a polyol, acarbohydrate and a proteinaceous material. For example, the matrix mayhave a pH of from about 5.0 to about 7.0. Such a preservation matrix maybe capable of maintaining at least about 10⁶ viable cells for a periodof at least about 12 months in vitro. In other examples, such a matrixmaintains at least about 10⁷ viable cells for a period of at least about12 months in vitro, and more preferably, at least about 10⁸ viable cellsfor a period of at least about 12 months in vitro. A preservation matrixmay be comprised of ingredients to minimize the damaging effectsencountered during the preservation process and to provide functionalproperties. For example when a Lactobacillus strain of the presentdisclosure is added to a preservation matrix for preservation, it may beconverted from an actively growing metabolic state to a metabolicallyinactive state.

In formulations of the present disclosure wherein a preservation matrixis contemplated, a biologically acceptable binding agent can be used toboth affix the bacterial culture or cultures to an inert carrier duringa preservation process and to provide protective effects (i.e.,maintains cell viability) throughout preservation and storage of thebacterial cells.

Antioxidants included in a preservation matrix may be provided to retardoxidative damage to the bacterial cells during the preservation andstorage process.

Polyols (i.e., polyhydric alcohols) included in a preservation matrixmay be provided to maintain the native, uncollapsed state of cellularproteins and membranes during the preservation and storage process. Inparticular, polyols interact with the cell membrane and provide supportduring the dehydration portion of the preservation process.

Carbohydrates included in a preservation matrix may be provided tomaintain the native, uncollapsed state of cellular proteins andmembranes during the preservation and storage process. In particular,carbohydrates provide cell wall integrity during the dehydration portionof the preservation process.

A proteinaceous material included in a preservation matrix may providefurther protection of the bacterial cell during the dehydration portionof the preservation process. Examples of the proteinaceous materialsinclude, but are not limited to skim milk and albumin.

One example of a method of preserving bacterial cells within apreservation matrix includes coating the cell matrix suspension onto aninert carrier that preferably is a maltodextrin bead. The coated beadscan then be dried, preferably by a fluid bed drying method. The coatedmaltodextrin beads can be stored as a powder, placed into gelatincapsules, or pressed into tablets.

In other formulations of the disclosure, the combinations of strains ofbacteria contemplated to be cultured can be formulated as a hard gelatincapsule.

In certain applications, the bacteria cultured with the methodsdescribed herein may be placed in a microencapsulation formulation. Suchmicroencapsulation formulations may have applicability for example inadministration to subjects via oral, nasal, rectal, vaginal or urethralroutes. Spray drying is the most commonly used microencapsulation methodin the food industry as it is economical, flexible and produces a goodquality product. The process involves the dispersion of the corematerial into a polymer solution, forming an emulsion or dispersion,followed by homogenization of the liquid, then atomization of themixture into the drying chamber. This leads to evaporation of thesolvent (water) and hence the formation of matrix type microcapsules.

The drying process is carried out in such a manner that a low residualmoisture content is present in the dry material. The percentage watercontent is preferably from about 2 to 3% by weight. This may be achievedby adding a post-drying step subsequent to the spray-drying step. Thedrying material for this purpose is, for example, post-dried in afluidized bed.

Instead of the above-described physical post-drying processes,desiccants can also be added to the dry material obtained from thespray-drying.

The content of viable microorganisms is in the range of from about 5×10⁵to 1×10¹² cfu/g of dry matter. These preparations are also referred toas powder concentrates.

Some end uses require fewer viable microorganisms and are thereforeblended to a lower final concentration by mixing the bacteria withlarger proportions of inert carrier material.

Some bacteria can survive environmental stresses through the formationof spores. This complex developmental process is often initiated inresponse to nutrient deprivation. It allows the bacteria to produce adormant and highly resistant cell. Spores can survive environmentalassaults that would normally kill other bacteria. Some stresses thatendospores can withstand include exposure to high temperatures, high UVirradiation, desiccation, chemical damage and enzymatic destruction. Theextraordinary resistance properties of endospores make them ofparticular importance because they are not readily killed by manyantibacterial treatments. Common bacteria that form spores includespecies from the Bacillus and Clostridium genera. Spores formed by thesebacteria remain in their dormant state until the spores are exposed toconditions favorable for growth. Spores of the specified bacteria can bebeneficially utilized in the disclosed compositions because of theirability to withstand processing methods and can have extended shelf lifeviabilities. Additionally, bacterial spores can require less processingbecause they do not require additional steps for preservation such asfreeze drying, spray drying, freezing and the like.

The disclosed composition can be fed to ruminal fermentors to reducescours events, improve animal health and animal productivity. Ruminalfermentors that can benefit from the present disclosure include but arenot limited to: cattle, sheep, goats, camels, llama, bison, buffalo,deer, wildebeest, antelope, and any other pre-gastric fermentor.Alternatively, the composition can be fed to cecal fermentors to reducescours events, improve animal health and animal productivity. Cecalfermentors that can benefit from the present disclosure include but arenot limited to: horses, ponies, elephants, rabbits, hamsters, rats,hyraxes, guinea pigs, and any other post-gastric fermentor that usingthe cecum as the primary location of fermentative digestion. Thecomposition can also be fed to intestinal fermentors to reduce scoursevents, improve animal health and animal productivity. Intestinalfermentors that can benefit from said disclosure include but are notlimited to: humans, pigs, chickens, and other post-gastric fermentorusing the large intestine as the primary location of fermentativedigestion.

The amount of bacteria administered to the animal feed can be any amountsufficient to achieve the desired increase in animal efficiency and/oranimal health. This amount can be anywhere from 1 to 10¹³ organisms perkg of animal feed. For example, amounts of about 10⁴ cfu/gram feed,about 5×10⁴ cfu/gram feed, about 10⁵ cfu/gram feed, about 5×10⁵ cfu/gramfeed, or ranges between 1 to 10¹³ organisms per kg of animal feed can beused. In some embodiments, the dried biological (bacteria) may beadministered to an animal through a variety of means including, but notlimited to, being distributed in an aqueous solution and subsequentlybeing applied to animal feed, water source, or directly fed to theanimal, or through direct application of the product onto animal feed ordirect administration or consumption by the animal.

In certain examples of the instant composition, the bacteria and methodsof the present disclosure involve two or more bacteria. In certainexamples, at least some of the bacteria are lactic acid-producingbacteria. These compositions would be provided in a combined amounteffective to achieve the desired effect, for example, the killing orgrowth inhibition of a pathogenic microorganism. This process mayinvolve administering different strains or species of lactic acidproducing microorganisms at the same time. In certain embodiments thedifferent strains or species may be combined into a single formulationfor administration. In other embodiments, the different strains orspecies of lactic acid producing microorganisms may be each in a singleformulation for administration. Still in other embodiments, some lacticacid producing microorganism strains or species may be combined into asingle formulation and others may be combined into a differentformulation.

When more than one inhibiting strain is included in the composition, theseveral bacterial components may be administered to the animal at thesame time or in a sequence sufficiently close together to instillsimilar effects in the animal as when the bacteria are administered atthe same time. When serially administered, it is contemplated that theseparate formulations can be administered within about 12-24 hours ofeach other and, more preferably, within about 6-12 hours of each other.In some situations, it may be desirable to extend the time period foradministration significantly such that days or weeks can lapse betweenthe respective administrations.

Various combinations may be employed, for example a formulationcontaining two species of lactic acid producing microorganisms is “A”and a second formulation containing three species of lactic acidproducing microorganisms is “B.”

In such embodiments, the administration may be, for example as such:A/B/A, B/A/B, B/B/A, A/B/B, A/B/B, B/A/A, A/B/B/B, B/A/B/B, B/B/B/A,B/B/A/B, A/A/B/B, A/B/A/B, A/B/B/A, B/B/A/A, B/A/B/A, B/A/A/B, A/A/A/B,B/A/A/A, A/B/A/A or A/A/B/A. It is further contemplated that otheradministrations may be used with three or more different formulations oflactic acid producing microorganisms.

In one example, the composition is designed for continual (once daily,for example) or periodic administration to ruminal fermentors throughouta feeding period in order to reduce the incidence and severity ofdiarrhea and/or improve overall health and/or inhibit pathogensassociated with the animal. In this embodiment, the compositioncomprises a mixture of probiotic bacteria supplemented with prebioticsubstances that can be introduced into the rumen and intestines of aruminal fermentor.

In another example, the composition is designed for continual orperiodic administration to cecal fermentors throughout feeding period inorder to reduce the incidence and severity of diarrhea and/or improveoverall health and/or inhibit pathogens associated with the animal. Inthis embodiment, the composition comprises a mixture of probioticbacteria supplemented with prebiotic substances that can be introducedinto the cecum and intestines of a cecal fermentor.

In yet another example, the novel composition is designed for continualor periodic administration to intestinal fermentors throughout thefeeding period in order to reduce the incidence and severity of diarrheaand/or improve overall health and/or inhibit pathogens associated withthe animal. In this embodiment, the composition comprises a mixture ofprobiotic bacteria supplemented with prebiotic substances that can beintroduced into the intestines of an intestinal fermentor.

A wide range of pathogenic bacteria can be inhibited or eliminatedthrough the disclosed compositions of bacteria such as lactic acidproducing probiotic bacteria. Specific examples of infectious diseasesor conditions of animals which can be caused by pathogenic bacteriainclude, but are not limited to: staphylococcal infections (caused, forexample, by Staphylococcus aureus, Staphylococcus epidermis, orStaphylococcus saprophyticus), streptococcal infections (caused, forexample, by Streptococcus pyogenes, Streptococcus pneumoniae, orStreptococcus agalactiae), enterococcal infections (caused, for example,by Enterococcus faecalis) diphtheria (caused, for example, byCorynebacterium diptheriae), anthrax (caused, for example, by Bacillusanthracis), listeriosis (caused, for example, by Listeriamonocytogenes), gangrene (caused, for example, by Clostridiumperfringens), tetanus (caused, for example, by Clostridium tetanus),botulism (caused, for example, by Clostridium botulinum), toxicenterocolitis (caused, for example, by Clostridium difficile), bacterialmeningitis (caused, for example, by Neisseria meningitidis), bacteremia(caused, for example, by Neisseria gonorrhoeae), E. coli infections(colibacilliocis), including urinary tract infections and intestinalinfections, shigellosis (caused, for example, by Shigella species),salmonellosis (caused, for example, by Salmonella species), Yersiniainfections (caused, for example, by Yersinia pestis, Yersiniapseudotuberculosis, or Yersinia enterocolitica), cholera (caused, forexample, by Vibrio cholerae), campylobacteriosis (caused, for example,by Campylobacter jejuni or Campylobacter fetus), gastritis (caused, forexample, by Helicobacter pylori), pseudomonas infections (caused, forexample, by Pseudomonas aeruginosa or Pseudomonas mallei), Haemophilusinfluenzae type B (HIB) meningitis, HIB acute epiglottitis, or HIBcellulitis (caused, for example, by Haemophilus influenzae), pertussis(caused, for example, by Bordetella pertussis), mycoplasma pneumonia(caused, for example, by Mycoplasma pneumoniae), nongonococcalurethritis (caused, for example, by Ureaplasma urealyticum),legionellosis (caused, for example, by Legionella pneumophila),syphillis (caused, for example, by Treponema pallidum), leptospirosis(caused, for example, by Leptospira interrogans), Lyme borreliosis(caused, for example, by Borrelia burgdorferi), tuberculosis (caused,for example, by Mycobacterium tuberculosis), leprosy (caused, forexample, by Mycobacterium leprae), actinomycosis (caused, for example,by Actinomyces species), nocardiosis (caused, for example, by Nocardiaspecies), chlamydia (caused, for example, by Chlamydia psittaci,Chlamydia trachomatis, or Chlamydia pneumoniae), Rickettsial diseases,including spotted fever (caused, for example, by Rickettsia ricketsii)and Rickettsial pox (caused, for example, by Rickettsia akari), typhus(caused, for example, by Rickettsia prowazekii), brucellosis (caused,for example, by Brucella abortus, Brucella melitens, or Brucella suis),and tularemia (caused, for example, by Francisella tularensis).

The present patent application claims priority to and is a continuationof each of the following patent applications previously filed on Dec.18, 2014, the disclosures of which are hereby expressly incorporatedherein in their entireties for purposes of present disclosure: Ser. Nos.14/575,916; 14/575,949; 14/575,974; 14/575,992 and 14/576,034.

The invention claimed is:
 1. A composition comprising a mixture havingequal viable numbers of each of the following four bacteria:Lactobacillus animalis strain MB101 having ATCC Accession NumberPTA-121710; Lactobacillus animalis strain MB102 having ATCC AccessionNumber PTA-121711; Enterococcus faecium strain MB505 having ATCCAccession Number PTA-121709; and Pediococcus acidilactici strain MB902having ATCC Accession Number PTA-121712; and wherein the mixture has anE. coli 0157:H7 growth inhibition rate of at least 93.1% when mixed withan equal number of viable E. coli 0157:H7 bacteria relative to the totalnumber of viable bacteria comprised by the four bacteria of the mixture.2. The composition recited in claim 1 wherein the mixture has aSalmonella typhimurium growth inhibition rate of at least 97.4% whenmixed with an equal number of viable Salmonella typhimurium bacteriarelative to the total number of viable bacteria comprised by the fourbacteria of the mixture.
 3. The composition recited in claim 2 whereinthe mixture has a Salmonella enteriditis growth inhibition rate of atleast 96.0% when mixed with an equal number of viable Salmonellaenteriditis bacteria relative to the total number of viable bacteriacomprised by the four bacteria of the mixture.
 4. The compositionrecited in claim 1 wherein the mixture has a Salmonella enteriditisgrowth inhibition rate of at least 96.0% when mixed with an equal numberof viable Salmonella enteriditis bacteria relative to the total numberof viable bacteria comprised by the four bacteria of the mixture.
 5. Thecomposition recited in claim 1 wherein the mixture is a growth inhibitorto the non-O157 STEC, Big-Six Escherichia coli strains comprising E.coli O121:H19; E. coli O45:H2; E. coli O103:H11; E. coli O145:NM, E.coli O26:H11; and E. coli O111.
 6. The composition recited in claim 1further comprises a carrier.
 7. The composition recited in claim 5further comprises a carrier and is packaged such that viable bacterianumber from about 5×10⁵ to 1×10¹² cfu/gram of packaged dry matter. 8.The composition recited in claim 6 wherein the carrier further comprisesat least one prebiotic chosen from the group comprising: afructo-oligosaccharides (FOS), inulin, isomalto-oligosaccharide,gentio-oligosaccharide, lactilol, lactosucrose, lactulose, xylosucrose,glycosylsucrose, pyrodextrin, soybean oligosaccharide,galacto-oligosaccharide (GOS), transgalactose-oligosaccharides,xylo-oligosaccharide (XOS), Malto-oligosaccharide (MOS), galactomannan,pentosan, beta-glucan, arabinan, galactan, palantinose-oligosaccharides,gluco-oligosaccharides, cyclo-inulo-oligosaccharides,chito-oligosaccharide, agaro-oligosaccharide, neo-oligosaccharide,garo-oligosaccharide, pectin and pectic polysaccharide.